Photodynamic compositions, methods of making, and uses thereof

ABSTRACT

Provided herein are photodynamic compositions that can contain a natural polymer scaffold and a photosensitizer, where the photosensitizer can be covalently or non-covalently attached to the natural polymer scaffold. Also provided herein are structures and objects that can contain the photodynamic compositions. Further provided herein are methods of making and using the photodynamic compositions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to co-pending U.S.Provisional Patent Application No. 62/334,150, filed on May 10, 2016,entitled “PHOTODYNAMIC COMPOSITIONS, METHODS OF MAKING, AND USESTHEREOF,” the contents of which is incorporated by reference herein inits entirety.

BACKGROUND

The survival of bacteria, fungi, and viruses on surfaces leads to thesubsequent transmission of these pathogens to new hosts, andsignificantly contributes to their proliferation, which in turnconsiderably increases their threat to human health, especially byantibiotic resistant strains. As such there exists an urgent need forantibacterial compositions and methods of use.

SUMMARY

In some aspects, provided herein are photodynamic compositions that cancontain a natural polymer scaffold; and a photosensitizer, wherein thephotosensitizer can be attached to the natural polymer scaffold. Thephotosensitizer can be attached to the natural polymer scaffold by acovalent bond. The photosensitizer can be attached to the naturalpolymer scaffold by a non-covalent bond. The non-covalent bond can be anelectrostatic interaction. The photosensitizer can be a positivelycharged photosensitizer. The positively charged photosensitizer can beselected from the group of: positively charged porphyrins, positivelycharged phthalocyanines, positively charged BODIPY based compounds,positively charged chlorins, positively charged bacteriochlorins,positively charged anthocyanins, positively charged rose Bengal,positively charged phenothiazine derivatives, and any permissiblecombinations thereof. The photosensitizer can be a negatively chargedphotosensitizer. The negatively charged photosensitizer can be selectedfrom the group of: negatively charged porphyrins (e.g.,5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin,5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-Pd(II)), negativelycharged phthalocyanines (phthalocyanine tetrasulphonic acid), negativelycharged BODIPY based compounds, negatively charged chlorins, negativelycharged bacteriochlorins, negatively charged anthocyanins, negativelycharged rose Bengal, negatively charged methylene blue and relatedphenothiazine derivatives, and any permissible combinations thereof. Thenatural polymer scaffold can be selected from the group consisting of:nanofibrillated cellulose, nanocrystalline cellulose, cellulose fibers,starch, lignin, chitosan, nanofibrillated chitosan, poly-glucosamine,and any permissible combinations thereof. The natural polymer scaffoldcan be negatively functionalized. The natural polymer scaffold can bepositively functionalized. In some aspects, any of the compositionsprovided herein can further contain a synthetic polymer. The syntheticpolymer can be polyacrylonitrile. In some aspects, any of thecompositions provided herein can further contain a secondphotosensitizer, wherein the second photosensitizer can be attached tothe synthetic polymer by a covalent or non-covalent interaction. In someaspects, any of the compositions provided here can form a coating whenapplied to the surface of a material. In some aspects, the photodynamiccompositions provided herein can be antimicrobial.

In other aspects, provided herein are objects and/or structures that caninclude any composition as provided herein. In some aspects, thecompositions as provided herein can form a coating on a surface of theobject or the structure. In some aspects, the compositions as providedherein can be assimilated into the object or the structure. In someaspects, the object or the structure can be a textile. In some aspects,the object or the structure can be a container or packaging material. Insome aspects the object or the structure can be a wall, ceiling, orfloor.

In other aspects provided herein are methods of making the compositionsprovided herein. The methods can contain the step of mixing a naturalpolymer scaffold and a photosensitizer, wherein the step of mixing canform a non-covalent or a covalent interaction between the naturalpolymer scaffold and the photosensitizer.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the present disclosure will be readily appreciatedupon review of the detailed description of its various embodiments,described below, when taken in conjunction with the accompanyingdrawings.

FIGS. 1A-1B shows representative photograph (FIG. 1A) and micrographicimages (FIG. 1B) of one embodiment of a polyacrylonitrile-basedphotodynamic composition.

FIG. 2 shows a graph demonstrating an antibacterial effect of thepolyacrylonitrile-based photodynamic composition.

FIG. 3 shows a graph demonstrating an antiviral effect of thepolyacrylonitrile-based photodynamic composition.

FIG. 4 shows a photographic image of NFC-MB formed from the addition of30 mg MB to 30.0 g 0.5 wt % TEMPO-oxidized NFC in water.

FIGS. 5A-5D show images of the NFC-NeoCryl 80/20 material with 0.05 wt %of various photosensitizers and their relevant structures: none (FIG.5A); TMPyP-Zn (FIG. 5B); Methylene Blue (FIG. 5C); or BODIPY (FIG. 5D).

FIG. 6 shows a graph demonstrating the results from antiviralphotodynamic inactivation studies against vesicular stomatitis virus(VSV). The black and dark grey bars represent the number of PFU/mL forthe following illuminated samples: material-free control,photosensitizer-free NFC control, 0.1 wt % TMPyP in NFC-NeoCryl 80/20,and 0.1 wt % MB in NFC-NeoCryl 80/20. The illumination conditions wereas follows: 30 min, 400-700 nm, 65±5 mW/cm² (total fluence of 118J/cm²).

FIGS. 7A-7B show graphs demonstrating the results from Photodynamicinactivation studies employing 0.1 wt % photosensitizer in NFC-NeoCryl80/20. (FIG. 7A) Gram-positive species: methicillin-resistant S. aureus(MRSA) ATCC-44. (FIG. 7B) Gram-negative species: multidrug-resistant A.baumannii (MDRAB) ATCC-1605. For both panels, displayed are theilluminated (dark grey) conditions as the percent survival of the darkcontrol (black) for 0.1 wt % TMPyP in NFC-NeoCryl 80/20 and 0.1 wt % MBin NFC-NeoCryl 80/20. For both bacteria, the illumination conditionswere as follows: 30 min, 400-700 nm, 65±5 mW/cm² (total fluence of 118J/cm²).

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the disclosure. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the disclosure, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the disclosure.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of molecular biology, microbiology,nanotechnology, organic chemistry, biochemistry, botany and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

Definitions

As used herein, “copolymer” can be used to refer to a polymer chainhaving two or more different types of monomers with in that chain.“Copolymer” can include block copolymers (such as di-block and tri-blockcopolymers), statistical (random) copolymers, gradient copolymers, andthe like.

As used herein, “homopolymer” can be used to refer to a polymer chainhaving only one type of monomer.

As used herein, “polymer” can refer to any type of polymer, includingbut not limited to homopolymers, copolymers, linear polymers, andbranched polymers.

As used herein, “photosensitizer” can refer to molecule, compounds, andcompositions that can produce a chemical, biochemical, and/or physicalchange in response to being stimulated with light. The light can be anywavelength of light including, but not limited to, all visible light,all UV light, infrared light, and fluorescent light.

As used herein, “photodynamic” refers to the ability of a compositionsto respond chemically, physically, biochemically, or otherwise change inresponse to being contacted or otherwise stimulated by a light. Thelight can be any wavelength of light including, but not limited to, allvisible light, all UV light, infrared light, and fluorescent light.

As used herein, “antimicrobial” can refer to the ability of acomposition to kill, eliminate, a microbe or population thereof.“Antimicrobial” can also refer to the ability of a composition toprevent the growth of a microbe or population thereof. “Antimicrobial”therefore can encompass the terms antibacterial, antiviral, antifungal,antiparasitic, bacteriostatic, and the like.

As used herein, the term “microbe” can refer to any living organism thatis too small to be observed by the naked human eye. As such, “microbe”includes but is not limited to bacteria, protists, some fungi, andviruses.

The term “molecular weight”, as used herein, generally refers to themass or average mass of a material. If a polymer or oligomer, themolecular weight can refer to the relative average chain length orrelative chain mass of the bulk polymer. In practice, the molecularweight of polymers and oligomers can be estimated or characterized invarious ways including gel permeation chromatography (GPC) or capillaryviscometry. GPC molecular weights are reported as the weight-averagemolecular weight (M_(w)) as opposed to the number-average molecularweight (M_(n)). Capillary viscometry provides estimates of molecularweight as the inherent viscosity determined from a dilute polymersolution using a particular set of concentration, temperature, andsolvent conditions.

As used herein “biodegradable” generally refers to a material that willdegrade or erode under physiologic conditions to smaller units orchemical species that are capable of being metabolized, eliminated, orexcreted by the subject. The degradation time is a function ofcomposition and morphology. Degradation times can be from hours toweeks.

The term “hydrophilic”, as used herein, refers to substances that havestrongly polar groups that readily interact with water.

The term “hydrophobic”, as used herein, refers to substances that lackan affinity for water; tending to repel and not absorb water as well asnot dissolve in or mix with water.

The term “lipophilic”, as used herein, refers to compounds having anaffinity for lipids.

The term “amphiphilic”, as used herein, refers to a molecule combininghydrophilic and lipophilic (hydrophobic) properties.

As used herein, “about,” “approximately,” and the like, when used inconnection with a numerical variable, generally refers to the value ofthe variable and to all values of the variable that are within theexperimental error (e.g., within the 95% confidence interval for themean) or within +/−10% of the indicated value, whichever is greater.

As used herein, “control” is an alternative subject or sample used in anexperiment for comparison purpose and included to minimize ordistinguish the effect of variables other than an independent variable.

As used herein, “positive control” refers to a “control” that isdesigned to produce the desired result, provided that all reagents arefunctioning properly and that the experiment is properly conducted.

As used herein, “negative control” refers to a “control” that isdesigned to produce no effect or result, provided that all reagents arefunctioning properly and that the experiment is properly conducted.Other terms that are interchangeable with “negative control” include“sham,” “placebo,” and “mock.”

DISCUSSION

According to the CDC's Healthcare-Associated Infections (HAI) PrevalenceSurvey, there were an estimated 722,000 HAIs in U.S. acute carehospitals in 2011, equivalent to about 1 out of every 25 inpatientshaving at least one health care-associated infection on any given day.Approximately 75,000 deaths were attributed to these infections, orabout 10% of the total HAIs. When one considers that these estimates ofthe national burden of health care-associated infections were limited toacute care hospitals, factoring in the magnitude of HAIs attributed toother settings (e.g., skilled nursing facilities, outpatient clinics,urgent care facilities) further highlights the scope and staggering costof nosocomial infections. One of the main contributing factors to HAIsis the ability of pathogens such as bacteria, fungi, and viruses toadhere to, and survive on, surfaces that leads to their subsequenttransmission to new hosts. As an example, Staphylococcus aureus cansurvive for weeks to months under dry conditions on the cotton andpolyester fabrics used in hospitals. Though typically not a concern forhealthy individuals, a second factor that contributes to HAIs is drugresistance, and five classes of antibiotic-resistant pathogens inparticular have emerged as major public health threats:vancomycin-resistant enterococci (VRE), methicillin-resistantStaphylococcus aureus (MRSA), multidrug-resistant mycobacteria,Gram-negative bacteria, and fungi. To combat these contributing factorsto HAIs, more research into effective surface disinfection andalternative materials (fabrics, plastics or coatings) with antimicrobialproperties capable of overcoming drug-resistance is needed. Moreover,food processing, packaging and service industries, waste watertreatment, daycare facilities, and personal households are other areaswhere infectious agents are easily spread, but may be countered by ananti-infective coating.

Several classes of antimicrobial agents are currently being investigatedor are commercially available, yet have disadvantages such as the lossof antimicrobial activity by leaching of the biocide, consumption of thegermicidal ability, environmentally hazardous agents, dependency ondirect contact of the antimicrobial entity with the microorganism,and/or their efficacy is often limited to a single class of microbe(i.e., only bacteria or fungi).

A promising area that circumvents many of these disadvantages while atthe same time satisfies many of the aforementioned criteria of an idealanti-infective material is antimicrobial photodynamic inactivation(aPDI). This branch of photomedicine employs light, air, and aphotosensitizer (PS) to generate primarily singlet oxygen (¹O₂) as thebiocidal agent, and represents a complementary strategy for thetreatment of microbial infections. Advantages of materials-based aPDIinclude i) employing singlet oxygen as the biocidal agent (which, givenits short lifetime and decay to harmless oxygen as an end product, canbe considered environmentally benign), ii) multiple routes to PSincorporation, including the attachment of the PS through electrostaticinteractions, encapsulation within a polymeric matrix, or directattachment via a covalent bond (prevents leaching into the environment),iii) the ability of the PS to potentially function in the absence ofdirect contact with the pathogen due to the diffusibility of singletoxygen, and iv) of great importance with respect to nosocomialinfections is that singlet oxygen or other photo-generated reactiveoxygen species cause non-specific damage from which microbial resistanceis unlikely to arise. To this latter point, aPDI has been shown topossess broad-spectrum antibacterial, antiviral, antifungal, andantiparasitic properties. Finally, as aPDI employs harmless white light,it has the advantage over Ultra Violet-C light irradiation (as anexample of another light-based sterilization technique), vaporizedhydrogen peroxide, or chlorine dioxide in that it can function withoutthe need for protecting people against the deleterious effects of thebiocidal agent.

A number of materials based upon a photodynamic mode of action have beenrecently reported. These include: synthetic polymer materials(polyurethane, polystyrene, polycaprolactone and polyamide-6) withencapsulated photosensitizers [e.g., free-base or zinctetraphenylporphyrin, zinc phthalocyanine, cationic5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP)] thatexhibit photobactericidal (E. coli) and photovirucidal (againstnon-enveloped polyomavirus and enveloped baculovirus) efficacy, as wellas natural polymer materials based on cellulose nanocrystals(Por⁽⁺⁾-CNCs), cellulose fibers (Por⁽⁺⁾-paper), or cotton fabrics thatpossess broad anti-infective efficacy against both bacteria (e.g.,Staphylococcus aureus, vancomycin-resistant Enterococcus faecium,Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiellapneumonia) and viruses (e.g., dengue-1, influenza A, and humanadenovirus-5).

However, these materials are not without limitations. First, most aresynthetic materials. Synthetic polymer materials have an advantage inthat their nanofibers are able to encapsulate photosensitizers,obviating the need for a covalent synthetic strategy, but their use ishindered by the fact that such non-natural scaffolds are not inherentlybiodegradable or biocompatible. Further, those that are not synthetic(e.g. cellulose fibers and cellulose nanocrystals) are not able toencapsulate photosensitizers, necessitating the need for tediouscovalent attachment/grafting of the photosensitizers onto the cellulosepolymer, thus limiting their scale-up potential and their practicalutility.

With that said, described herein are describe are photodynamiccompositions that can be antimicrobial and methods of using thephotodynamic compositions. The photodynamic compositions can include anatural polymer scaffold and an attached photosensitizer. Thephotodynamic compositions provided herein can have broad-spectrumantibacterial, antiviral, antifungal, and/or antiparasitic properties.The photodynamic compositions provided herein can also have theadvantage in that can utilize white light. As such, they can functionwithout the need for protecting people and/or animals from a deleteriouseffect of the biocidal agent. The compositions provided herein can beused as coatings that can be applied to a material, incorporated intomaterials and/or textiles that can have further use in variousindustries.

Other compositions, compounds, methods, features, and advantages of thepresent disclosure will be or become apparent to one having ordinaryskill in the art upon examination of the following drawings, detaileddescription, and examples. It is intended that all such additionalcompositions, compounds, methods, features, and advantages be includedwithin this description, and be within the scope of the presentdisclosure.

Photodynamic Compositions

A number of materials based upon a photodynamic mode of action have beenrecently reported. These include: synthetic polymer materials(polyurethane, polystyrene, polycaprolactone and polyamide-6) withencapsulated photosensitizers [e.g., free-base or zinctetraphenylporphyrin, zinc phthalocyanine, cationic5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrin (TMPyP)] thatexhibit photobactericidal (E. coli) and photovirucidal (againstnon-enveloped polyomavirus and enveloped baculovirus) efficacy, as wellas natural polymer materials based on cellulose nanocrystals(Por⁽⁺⁾-CNCs), cellulose fibers (Por⁽⁺⁾-paper), or cotton fabrics thatpossess broad anti-infective efficacy against both bacteria (e.g.,Staphylococcus aureus, vancomycin-resistant Enterococcus faecium,Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiellapneumonia) and viruses (e.g., dengue-1, influenza A, and humanadenovirus-5).

Despite the existence of these materials, NFC-PS can be distinguishedfrom these existing compositions. In some aspects, NFC-PS incorporatesnanofibrillated cellulose (NFC) scaffold, which has not been reported tobe used in antimicrobial materials possibly due to its relatively recentorigins compared with cellulose fibers and nanocrystals. Moreover, therelative lack of studies on NFC properties/chemistry in comparison tocellulose nanocrystals and fibers, coupled with the difficulty in makingNFC in appreciable quantities that are of a uniform consistency andquality, make NFC a non-obvious choice for a cellulose-based (or naturalpolymer) scaffold. Finally, previous cellulose-based materials employeda covalent attachment or grafting of the photosensitizer to thecellulose scaffold; here, due to the unique size of cellulosenanofibers, NFC can utilize the nanofiber encapsulation strategysuccessfully employed with synthetic nanofibers, obviating the need fora tedious covalent synthetic strategy and counterintuitively making NFCmore amenable to scale-up in what is a non-obvious approach forcellulose that is unique to NFC.

With the limitations of currently available compositions in mind,described herein are photodynamic compositions that can include anatural polymer scaffold and a photosensitizer, where thephotosensitizer can be attached to the natural polymer scaffold. In someembodiments, the photosensitizer can be attached to the natural polymerscaffold by a non-covalent bond. In some embodiments, the non-covalentbond is an electrostatic interaction. In some embodiments thephotosensitizer can be attached to the natural polymer scaffold by acovalent bond. The photodynamic compositions provided herein can bebiodegradable and/or biocompatible.

The photodynamic compositions can be formulated as a sprayable liquidthat can be applied to a coating to a surface of an object or astructure. The photodynamic compositions can be formulated such that thecomposition can be assimilated into an object or a structure. Thephotodynamic compositions described herein can be formulated as a 3Dprinting ink. As such, also provided herein are objects and structuresthat can have a surface coating of a photodynamic composition providedherein. Also provided are objects and structures that have aphotodynamic composition that is assimilated therein.

Natural Polymer Scaffolds

The photodynamic composition can contain one or more natural polymerscaffolds. The natural polymer scaffold(s) can contain nanofibrillatedcellulose, nanocrystalline cellulose, cellulose fibers, starch, lignin,chitosan, nanofibrillated chitosan, poly-glucosamine, and/or anypermissible combinations thereof. The natural polymer scaffolds can haveany molecular weight needed, for example, to obtain the desiredproperties of the photodynamic composition. The natural polymer scaffoldcan have a molecular weight from about 100 Da to 100,000 kDa or more.The natural polymer scaffold can be included in the photodynamiccomposition at about 0.001 wt % to about 99.9 wt % of the totalphotodynamic composition. The natural polymer scaffold can have a netpositive charge, a net negative charge, or a net neutral charge. Thesurface charge density of the natural polymer scaffold can range fromabout 0 to about +1 charge per D-glucose monomer for the positivelycharged scaffold, or range from about 0 to −1 charger per D-glucosemonomer for the negatively charged scaffold. In some embodiments wherethe natural polymer scaffold can be or can include nanofibrillatedcellulose, the hydrophobic to hydrophilic ratio can vary with wettingcontact angles ranging from 0<θ<180°.

The natural polymer scaffold can further include an additive. Suitableadditives can include, but are not limited to, acrylic polymers, acrylicco-polymers, acrylic emulsion polymers, acrylic co-polymer emulsions. Insome aspects the additive is NeoCryl XK-98. Other additives can include,but not limited to, pigments, stabilizers, driers, thickeners,preservatives, dispersants, silicones, thixotropic agents,photoinitiators for light curable coatings (e.g. camphorquinone), andanti-settling agents. In some aspects, the additive can surface modifythe natural polymer scaffold as described below. The additive can beincorporated with the natural polymer scaffold at a ratio of naturalpolymer scaffold:additive (wt:wt based upon dry solids) that can rangefrom about 51:49 to about 99.9:0.1 and any range in between. In someaspects, the additive can be incorporated with the natural polymerscaffold at a ratio of natural polymer scaffold:additive (wt:wt basedupon dry solids) that can be about 51:49; 55:45; 60:40; 65:35; 70:30;75:25; 80:20; 85:15; 90:10; 95:5; 99;1.

In addition to the natural polymer scaffold described above, thephotodynamic composition can include an additional polymer, which may ormay not be the same as an additive. The additional polymer can benatural or synthetic. The additional polymer can polymerize with or beotherwise coupled to the natural polymer scaffold described above. Theadditional polymer can be included in the photodynamic composition at 0%to about 95% of the total photodynamic composition. The additionalpolymer can have a molecular weight from about 100 Da to 100,000 kDa ormore.

Suitable additional polymers include, but are not limited to: chitosan,natural rubber, lignin, poly-glucosamine, epoxy resins, cellulosicpolymers such as starch and polysaccharides; hydrophilic polypeptides;poly(amino acids) such as poly-L-glutamic acid (PGS), gamma-polyglutamicacid, poly-L-aspartic acid, poly-L-serine, or poly-L-lysine;polyalkylene glycols and polyalkylene oxides such as polyethylene glycol(PEG), polypropylene glycol (PPG), and poly(ethylene oxide) (PEO);poly(oxyethylated polyol); poly(olefinic alcohol);polyvinylpyrrolidone); poly(hydroxyalkylmethacrylamide);poly(hydroxyalkylmethacrylate); poly(saccharides); poly(hydroxy acids);poly(vinyl alcohol), and copolymers thereof; polyhydroxy acids such aspoly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolicacids); polyhydroxyalkanoates such as poly3-hydroxybutyrate orpoly4-hydroxybutyrate; polycaprolactones; poly(orthoesters);polyanhydrides; poly(phosphazenes); poly(lactide-co-caprolactones);polycarbonates such as tyrosine polycarbonates; polyamides (includingsynthetic and natural polyamides), polypeptides, and poly(amino acids);polyesteramides; polyesters; poly(dioxanones); poly(alkylene alkylates);hydrophobic polyethers; polyurethanes; polyetheresters; polyacetals;polycyanoacrylates; polyacrylates; polymethylmethacrylates;polysiloxanes; poly(oxyethylene)/poly(oxypropylene) copolymers;polyketals; polyphosphates; polyhydroxyvalerates; polyalkylene oxalates;polyalkylene succinates; poly(maleic acids), as well as copolymersthereof. In certain embodiments, the hydrophobic polymer is an aliphaticpolyester; polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terephthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andcopolymers thereof, alkyl cellulose such as methyl cellulose and ethylcellulose, hydroxyalkyl celluloses such as hydroxypropyl cellulose,hydroxy-propyl methyl cellulose, and hydroxybutyl methyl cellulose,cellulose ethers, cellulose esters, nitro celluloses, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, carboxylmethyl cellulose, cellulosetriacetate, cellulose sulphate sodium salt, polymers of acrylic andmethacrylic esters such as poly (methyl methacrylate),poly(ethylmethacrylate), poly(butylmethacrylate),poly(isobutylmethacrylate), poly(hexylmethacrylate),poly(isodecylmethacrylate), poly(lauryl methacrylate), poly (phenylmethacrylate), poly(methyl acrylate), poly(isopropyl acrylate),poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,polypropylene poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), poly(vinyl acetate, poly vinylchloride polystyrene and polyvinylpyrrolidone, derivatives thereof,linear and branched copolymers and block copolymers thereof, and blendsthereof. Exemplary biodegradable polymers include polyesters, poly(orthoesters), poly(ethylene imines), poly(caprolactones),poly(hydroxyalkanoates), poly(hydroxyvalerates), polyanhydrides,poly(acrylic acids), polyglycolides, poly(urethanes), polycarbonates,polyphosphate esters, polyphosphazenes, derivatives thereof, linear andbranched copolymers and block copolymers thereof, and blends thereof.

Additional photosensitizers can be attached to the additional polymers.The additional photosensitizer can be attached via a covalent or anon-covalent bond to the additional polymers. The additionalphotosensitizers can be cationic, anionic, or neutral. Suitablephotosensitizers are described elsewhere herein.

The natural polymer scaffold can be surface modified. The naturalpolymer scaffold can be surface modified to be cationic and/or at leastcontain more positively charged moieties than a non-surface modifiednatural polymer scaffold. Natural polymer scaffolds that are surfacemodified to be cationic and/or at least contain more positively chargedmoieties than a non-surface modified natural polymer scaffold can havean increased and/or strengthened electrostatic interaction between ananionic photosensitizer and the natural polymer scaffold as compared toa non-surface modified natural polymer scaffold. The natural polymerscaffold can be surface modified to be anionic and/or at least containmore negatively charged reactive groups than a non-surface modifiednatural polymer scaffold. Natural polymer scaffolds that are surfacemodified to be cationic and/or at least contain more negatively chargedmoieties than a non-surface modified natural polymer scaffold can havean increased and/or strengthened electrostatic interaction between acationic photosensitizer and the natural polymer scaffold as compared toa non-surface modified natural polymer scaffold.

Photosensitizers

The photodynamic compositions provided herein can also include one ormore photosensitizers. The photosensitizer can be attached to thenatural polymeric scaffold. In some embodiments the photosensitizer canbe attached to the natural polymeric scaffold by a non-covalent bond.The non-covalent bond can be an electrostatic interaction, ahydrogen-bonding interaction, or a hydrophobic interaction. In someembodiments, the photosensitizer can be attached to the naturalpolymeric scaffold by a covalent bond. In some embodiments where morethan one photosensitizer is attached to the natural polymeric scaffold,at least one photosensitizer can be non-covalently attached to thenatural polymeric scaffold and at least one photosensitizer can becovalently attached to the natural polymeric scaffold. Thephotosensitizer can be included in the photodynamic composition at about0.001 wt % to about 50 wt % and any amount or range of amounts inbetween. In some aspects, the photosensitizer can be incorporated atabout 0.01 to about 0.1 wt % and any amount or range of amounts inbetween. In some aspects the photosensitizer can be incorporated atabout 0.05 wt %.

The photosensitizer can be positively charged. Suitable positivelycharged photosensitizers include, but are not limited to: positivelycharged porphyrins (e.g. Por⁽⁺⁾,5,10,15,20-tetrakis(1-methyl-4-pyridinyl)porphyrin tetratosylate,5,10,15,20-tetrakis(4-N,N,N-trimethylanilinium)porphyrin tetrachloride),positively charged phthalocyanines, positively charged BODIPY basedcompounds (e.g., DIMPy-BODIPY), positively charged chlorins, positivelycharged bacteriochlorins, positively charged anthocyanins, positivelycharged rose Bengal, positively charged phenothiazine derivatives (e.g.,methylene blue), positively charged metallated derivatives (e.g.TMPyP-Zn), and any permissible combinations thereof.

The photosensitizer can be negatively charged. Suitable negativelycharged photosensitizers include, but are not limited to: negativelycharged porphyrins (e.g.,5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin,5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin-Pd(II)), negativelycharged phthalocyanines (phthalocyanine tetrasulphonic acid), negativelycharged BODIPY based compounds, negatively charged chlorins, negativelycharged bacteriochlorins, negatively charged anthocyanins, negativelycharged rose Bengal, negatively charged methylene blue and relatedphenothiazine derivatives, negatively charged metallated derivatives,and any permissible combinations thereof.

The photosensitizer can be neutral. Suitable neutral photosensitizersinclude, but are not limited to: neutral porphyrins (e.g.,tetraphenylporphyrin), neutral phthalocyanines, neutral BODIPY basedcompounds (e.g., 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), neutralchlorins, neutral bacteriochlorins, neutral anthocyanins, neutral roseBengal (e.g. methyl ester of Rose Bengal), neutral phenothiazines,neutral metallated derivatives, and any permissible combinationsthereof.

Methods of Making the Photodynamic Compositions

Photodynamic compositions can be made by a covalent attachment of thephotosensitizer to the polymer, including but not limited toesterification, copper-catalyzed alkyne-azide cycloaddition reactions(“Click” chemistry, Huisgen cyclization), ether bond linkages, amidebond linkages, coupling reactions (e.g., Sonagashira, Kumada, Heck,Nigishi, Stille, Suzuki), and bioconjugations reactions (e.g.,amine-reactive succinimidyl esters/NHS-ester, cysteine-maleimide,cysteine-iodoacetamide, disulfide bond formation, tyrosine diazoniumsalt, native chemical ligation, Staudinger ligation, modification ofketones and aldehydes, N-terminal aldehyde). Photodynamic compositionsmay also be made by non-covalent attachments, including but not limitedto electrostatic attachment (e.g., ion-ion, ion-dipole interactions,dipole-dipole interactions/hydrogen-bonding), hydrophobic interactions,and physical encapsulations methods. Electrostatic ion-ion attachment ofan ionic photosensitizer with an ionic polymer includes positivelycharged photosensitizers that are mixed with polymers bearing negativelycharged functional groups, or negatively charged photosensitizers mixedwith polymers bearing positively charged functional groups.Electrostatic ion-dipole attachment includes positively or negativelycharged photosensitizers that are mixed with polymers possessingpermanent dipoles, or photosensitizers possessing permanent dipolesmixed with polymers bearing positively or negatively charged functionalgroups. Electrostatic dipole-dipole (e.g., hydrogen bonding) attachmentincludes photosensitizers possessing permanent dipoles that are mixedwith polymers possessing permanent dipoles. Hydrophobic interactionattachment includes hydrophobic (e.g., non-polar) photosensitizers thatare mixed with hydrophobic (e.g., non-polar) polymers. Physicalencapsulations methods include the physical trapping/caging of thephotosensitizer within the polymer matrix.

Methods of Using the Photodynamic Compositions

The photodynamic compositions described herein can have use in a widevariety of applications where microbial control, microbial reduction,disinfection and/or sterilization is desired. Generally speaking, thephotodynamic compositions provided herein can be coated onto orassimilated into a range of materials, thereby creating novel renewable,biodegradable, anti-infective consumer staples. The photodynamiccompositions can be applied as coating to a surface of an object or astructure. The photodynamic composition can be applied as a coating to asurface of an object or a structure by spraying, painting, or otherwisedepositing the photodynamic composition to the surface of the object orthe structure.

For example the photodynamic compositions provided herein can be used asa paint-like spray coating (walls, draperies, blinds) for pathogenreduction/antimicrobial applications in hospitals and related healthcaresettings; assimilated into textiles for sterile hospital gowns/masks,hospital linens, and patient garments; assimilated in seat fabrics,seatback trays, and other areas in high density transportation fields(airplanes, trains, subways, public restrooms), with further applicationin the space industry; blended and/or applied as a film to thepolycarbonate shells in neonatal intensive care units, indwellingcatheters, bandages for continuous light-activated sterilization ofwounds (particularly for burn victims), or for treatment of medicalwaste; applied as film to cover touchscreens for computers, airportcheck-in kiosks, and smart devices (phones, tablets, etc.); used as ananti-mold wallpaper for basements and crawl spaces; coated onto paper orplastics for antimicrobial packaging for food storage, safety,preparation, and handling; assimilated into, formed as, or applied as acoating to filters for flow-sterilization applications of municipalwater sources. In some instances, the photodynamic compositions providedherein can be applied to a surface of an object or a structure via 3-Dprinting.

EXAMPLES

Now having described the embodiments of the present disclosure, ingeneral, the following Examples describe some additional embodiments ofthe present disclosure. While embodiments of the present disclosure aredescribed in connection with the following examples and thecorresponding text and figures, there is no intent to limit embodimentsof the present disclosure to this description. On the contrary, theintent is to cover all alternatives, modifications, and equivalentsincluded within the spirit and scope of embodiments of the presentdisclosure.

Example 1

Recently, a photoactive material, termed Paper-por has been described.See Carpenter et al., Biomacromolecules. 2015. 16:2482-2492, theentirety of which is incorporated herein by reference as if expressed inits entirety. In the Paper-por material, a porphyrin-basedphotosensitizer Por(+) is covalently attached to cellulose fibers.Paper-por material was demonstrated to be effective againstStaphylococcus aureus, vancomycin-resistant Enterococcus faecium,Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiellapneumoniae, with inactivation of all bacterial strains studied by99.99+%. Paper-Por was also found to inactivate dengue-1 virus(>99.995%), influenza A (□99.5%), and human adenovirus-5 (□99%) all to ahigh level. As such, the Paper Por material demonstrates that cellulosefibers that are attached to a photosensitizer can be effective atkilling a large variety of microbes.

In this Example a simplified strategy of embedding photosensitizers intopolymers that does not rely on the covalent attachment of thephotosensitizer to the scaffold used in Paper-por is demonstrated. Morespecifically, this Example at least demonstrates using polyacrylonitrile(PAN) to make PAN-Por, an electrospun non-woven textile having anencapsulated photosensitizer (Por⁽⁺⁾).

Materials and Methods

Buffer salts were purchased from Fisher Scientific, Nutrient Broth#234000 was obtained from BD Difco, LB broth Miller from EMD Chemicals,and Tryptic Soy Broth from Teknova. Unless otherwise specified, allother chemicals were obtained from commercial sources in reagent gradepurity or better. Deionized water used for all media and buffers.UV-visible absorption measurements were performed on a Varian Cary 50Bio instrument or a Genesys 10 UV scanning spectrophotometer from ThermoElectron Corp for single wavelength measurements. The photosensitizerPor⁽⁺⁾ was synthesized as described previously (Feese, E.; Sadeghifar,H.; Gracz, H. S.; Argyropoulos, D. S.; Ghiladi, R. A. Photobactericidalporphyrin-cellulose nanocrystals: synthesis, characterization, andantimicrobial properties. Biomacromolecules 2011, 12, 3528-3539;Carpenter, B. L.; Feese, E.; Sadeghifar, H.; Argyropoulos, D. S.;Ghiladi, R. A. Porphyrin-cellulose nanocrystals: a photobactericidalmaterial that exhibits broad spectrum antimicrobial activity. Photochem.Photobiol. 2012, 88, 527-536; Feese, E., North Carolina StateUniversity, 2011).

Field-emission scanning electron microscopy (FE-SEM, FEI Verios 460L,USA) was performed at an acceleration voltage of 2 kV to observe themorphology of the obtained samples. Thermal gravimetric analysis (TGA)was carried out on a TA instrument TGAQ50 ramping 8° C./min under N₂purging.

Electrospinning of PAN-Por⁽⁺⁾:

Polyacrylonitrile (PAN, Mw=150,000) and N, N-dimethylformamide (DMF)were purchased from Sigma-Aldrich and used as received. The solution wasprepared by firstly dissolving PAN powder into DMF solvent with a weightpercentage of 5 wt. %. The PAN/DMF solution was stirred for over 24hours, followed by the addition of cationic porphyrin (1130 g mol⁻¹).The mass of cationic porphyrin was 10 wt. with respect to the mass ofPAN. The solution was further stirred for 24 hours prior toelectrospinning. A variable high voltage power supply (Gamma ES40P-20W/DAM) was used to provide a high voltage (15 kV) for electrospinning.The flow rate applied was 0.75 mL h⁻¹. The needle-to-collector distancewas set at 15 cm and electrospun fibers were collected on an aluminumfoil. Each sample of PAN-Por⁽⁺⁾ (˜1 mg) was then placed in a 24-wellplate and washed 8 times with a minimum of 2 mL deionized water perwashing, thereby removing adventitiously bound Por⁽⁺⁾ to a concentrationof less than 19 nM as determined by UV-visible spectroscopy.

Determination of PAN-Por⁽⁺⁾ Porphyrin Loading:

˜9 mg of dry, washed textile was dissolved in 4 mL dimethylformamide and6 mL deionized water, thereby fully solubilizing the Por⁽⁺⁾photosensitizer. The resulting solution was syringe filtered (0.22 μm)to remove any trace undissolved PAN nanofibers, diluted 1:6 withdeionized water, and the concentration of the Por⁽⁺⁾ was determined byUV-visible spectroscopy using ε_(Soret)=195,000 M⁻¹ cm⁻¹ (Ghiladi, R. A.Porphyrin-cellulose nanocrystals: a photobactericidal material thatexhibits broad spectrum antimicrobial activity. Photochem. Photobiol.2012, 88, 527-536; Feese, E., North Carolina State University, 2011).

Cell Culture:

All bacteria were grown in 5 mL cultures incubated at 37° C. on anorbital shaker at 500 RPM under the following growth conditions:methicillin susceptible Staphylococcus aureus 2913 was grown in trypticsoy broth without antibiotics; vancomycin resistant Enterococcus faecium(ATCC-2320) was grown in DB Difco Bacto Brain Heart Infusion 237500 with50 μg/mL ampicillin; Escherichia coli BL21-(Dε3)pLysS (Stratagene, USA)was grown in Miller LB media with 100 μg/mL ampicillin; Acinetobacterbaumannii (ATCC-19606) was grown in Miller LB media without antibiotics.Klebsiella pneumoniae (ATCC-2146) was grown in DB Difco Nutrient broth#234000 with 100 μg/mL ampicillin. Each bacterium was grown to aconcentration of 1-4×10⁸ CFU/mL (determined spectrophotometrically fromgrowth curves using a Genesys 10 UV scanning spectrophotometer) prior tobeing pelleted by centrifugation (15 min, ˜4150 g). Once pelleted, thesupernatant was decanted and the cells were resuspended in 5 mL of brothmedia and diluted to ˜10⁸ CFU mL⁻¹ (determined spectrophotometrically).

Viral Propagation:

Vesicular stomatitis virus (VSV) NJ strain was propagated on Vero cellsand titered by plaque assay on Vero cells. Human adenovirus-5 (HAd-5)was propagated on the human lung carcinoma cell line A549 and titered onthe same cells. Plaques were visualized by crystal violet staining.

Photodynamic Inactivation Assay:

All photosensitization experiments were performed using a non-coherentlight source, PDT light model LC122 (LumaCare, USA), equipped with a LUMV fiber optic probe (400-700 nm band pass filter, average transmittanceT_(avg) ˜95±3%) and an OSRAM Xenophot lamp model 64653 HLX (24 V, 250VV). The fluence rate was measured with an Orion power meter (OphirOptronics Ltd, Israel). All experiments were conducted in triplicate ata minimum, and statistical significance was assessed via a two-tailed,unpaired Student's t-test.

Bacteria:

a sterile 24-well plate (BD Falcon, flat bottom) was prepared withPAN-Por⁽⁺⁾ cut to precisely fit the well bottom (□1 cm diam.) using acustom hole punch. Aliquots (100 μL) of cell culture were transferred toeach well and illuminated with visible light (400-700 nm) with a fluencerate of 65±5 mW/cm² for a variable period of time (5-60 min) whilemagnetically stirred. Studies were repeated as described with PAN(PS-free material control), and in the absence of material (cells-onlycontrol), under both illuminated and non-illuminated (dark) conditions.After illumination, each well was 1:10 serially diluted five times. 10μL from the undiluted well and from each dilution, as well as from thedark control, were plated and incubated in the dark at 37° C. Eachbacterium was grown on gridded six column square agar plates made withtheir respective growth media without antibiotics. The survival rate wasdetermined from the ratio of CFU/mL of the illuminated solution versusthat of the dark control. The minimum detection limit was 100 CFU/mL(based on 10 μL plated from the 1 mL undiluted well). Variations in theconcentration of the starter culture (1-4×10⁸ CFU/mL) resulted in avariation of the detection limit spanning the region of 0.001-0.0001%survival. Samples with PAN-Por⁽⁺⁾ present but kept in the dark (darkcontrol) and illuminated samples of PAN without PS (light control)served as controls.

Vesicular Stomatitis Virus:

25 μl of a VSV stock (5×10⁸ plaque forming units (PFU)/ml) were added toeither empty well (Control), PAN or PAN-Por⁽⁺⁾ in wells of a 96 wellplate in the dark prior to 30 min under visible light illumination(400-700 nm; 65±5 mW/cm²). Control experiments were similarly performedin the dark. Treatments were performed in biological triplicates. Afterillumination 100 μL of MEM supplemented with 10 mM HEPES, 1% FBS andantibiotics were added to wash remaining viruses off the textiles. Virussamples were subsequently titered on Vero cells, and the virusconcentration was determined by plaque assay (detection limit of 40PFU/mL). Specifically, viruses were titered by serial 10-fold dilutionon Vero cells in 12-well plates at 37° C. Plaques were detected bycrystal violet staining 48 h after infection. Where virus wasdetectable, the plaques at dilutions where wells contained between 10-30plaques were counted for titer determination.

Human Adenovirus-5:

25 μl of a human adenovirus 5 stock (4.5×10⁷ PFU/ml) were added toeither empty well (Control), PAN or PAN-Por⁽⁺⁾ in wells of a 96 wellplate in the dark prior to 30 min under visible light illumination(400-700 nm; 65±5 mW/cm²). Treatments were performed in biologicaltriplicates. After illumination 100 μL of DMEM supplemented with 10% FBSand antibiotics were added to wash remaining viruses off the textiles.Control experiments were similarly performed in the dark. Virus wastitered by serial 10-fold dilution on A549 cells in 12-well plates at37° C. Plaques were detected by crystal violet staining 120 h afterinfection. Where virus was detectable, the plaques at dilutions wherewells contained between 10-40 plaques were counted for titerdetermination.

Results

PAN-Por⁽⁺⁾ (FIGS. 1A-1B) was observed to me more effective thanPaper-por at killing microbes. See FIGS. 2-3. It was observed thatPAN-Por⁽⁺⁾ yielded complete killing (about 100%) of E. coli, A.baumannii, and S. aureus, and 99.9995% reduction of K. pneumoniae and P.aeruginosa (FIG. 2). In other words, for all strains examined, whenstarting with 100 million pathogenic cells present prior to lightactivation, fewer than 500 cells remained viable after only 30 minutesof aPDI treatment. PAN-Por⁽⁺⁾ was also found to inactivate vesicularstomatitis virus (>99.9999%) (FIG. 3) and human adenovirus-5 (about99.5%) (data not shown).

Example 2

NFC-PS takes advantage of both approaches given above, namely combiningnanofibrillated cellulose (as a renewable, compostable, andbiodegradable biopolymer) with an embedded photosensitizer strategy(thereby minimizing synthesis and allowing for scale-up and the use ofcommercially available photosensitizers) to create an antimicrobialcoating that can be both easy to apply (sprayable, brushable, or can beincorporated into 3D printing technology) and effective against a widerange of pathogens, including bacteria, viruses, and fungi. As opposedto cellulose nanocrystals (Feese, E.; Sadeghifar, H.; Gracz, H. S.;Argyropoulos, D. S.; Ghiladi, R. A. Photobactericidalporphyrin-cellulose nanocrystals: synthesis, characterization, andantimicrobial properties. Biomacromolecules 2011, 12, 3528-3539;Carpenter, B. L.; Feese, E.; Sadeghifar, H.; Argyropoulos, D. S.;Ghiladi, R. A. Porphyrin-cellulose nanocrystals: a photobactericidalmaterial that exhibits broad spectrum antimicrobial activity. Photochem.Photobiol. 2012, 88, 527-536) or fibers (Carpenter, B. L.; Scholle, F.;Sadeghifar, H.; Francis, A. J.; Boltersdorf, J.; Weare, W. W.;Argyropoulos, D. S.; Maggard, P. A.; Ghiladi, R. A. Synthesis,Characterization, and Antimicrobial Efficacy of PhotomicrobicidalCellulose Paper. Biomacromolecules 2015, 16, 2482-2492), the use ofnanofibrillated cellulose (NFC) is due to its attractive high surfacearea characteristics whose gelatinous nature makes it amenable to beingreadily applied on the surfaces of materials. However, thisnanofibrillated cellulose is relatively new (as compared to cellulosefibers and cellulose nanocrystals) and its laborious and comparativelydifficult production (as compared to cellulose fibers and cellulosenanocrystals) has directed away from its use as a scaffold material inthis context. In this example a NFC-MB compositions was generated todemonstrate the feasibility of NFC as a scaffold material for use in aphotodynamic composition.

Materials and Methods

Preparation of NFC-MB:

To a suspension of 30.0 grams TEMPO-oxidized nanofibrillated cellulose(0.5 wt % in water) was added 30 mg methylene blue pre-dissolved in 5 mLwater. The mixture was stirred vigorously for 10 minutes, and thenpoured into a square petri dish. The water was allowed to evaporate atroom temperature for three days, after which NFC-MB formed as a thinfilm that was easily removed from the petri dish.

Photodynamic Inactivation Assay:

All photosensitization experiments were performed using a non-coherentlight source, PDT light model LC122 (LumaCare, USA), equipped with a LUMV fiber optic probe (400-700 nm band pass filter, average transmittanceT_(avg) ˜95±3%) and an OSRAM Xenophot lamp model 64653 HLX (24 V, 250VV). The fluence rate was measured with an Orion power meter (OphirOptronics Ltd, Israel).

Bacteria Testing:

Two pieces of NFC-MB were cut to precisely fit the bottom (□1 cm diam.)of a 24-well plate using a custom hole punch, and were illuminated asdescribed above under the Photodynamic Inactivation Assay. Each NFC-MBwas then swabbed with a sterile cotton applicator, and the cottontransferred to a culture tube into which bacterial culture medium wasadded (either as LB or TSB). After agitation, 10 μL from each culturetube was plated directly on gridded six column square agar plates madewith their respective growth media without antibiotics. The remainingculture solutions were incubated at 37° C. for one, and an additional 10μL from each culture tube was plated on a second set of gridded sixcolumn square agar plates. The plates were grown overnight at 37° C. andsubjected to colony counting the next day.

Viral Testing: Vesicular Stomatitis Virus:

25 μl of a VSV stock (5×10⁸ plaque forming units (PFU)/ml) were added toeither empty well (Control), or disks of NFC, NFC TMPyP or NFC MB) inwells of a 96 well plate in the dark prior to 30 min under visible lightillumination (400-700 nm; 65±5 mW/cm²). Control experiments weresimilarly performed in the dark. Treatments were performed in biologicaltriplicates. After illumination 100 μL of MEM supplemented with 10 mMHEPES, 1% FBS and antibiotics were added to wash remaining viruses offthe disks. Virus samples were subsequently titered on Vero cells, andthe virus concentration was determined by plaque assay (detection limitof 40 PFU/mL). Specifically, viruses were titered by serial 10-folddilution on Vero cells in 12-well plates at 37° C. Plaques were detectedby crystal violet staining 48 h after infection. Where virus wasdetectable, the plaques at dilutions where wells contained between 10-30plaques were counted for titer determination.

Results

The prepared NFC-MB is demonstrated in FIG. 4. The final composition ofthe material shown in FIG. 4 was 30 mg MB:150 mg TEMPO-oxidized NFC. Inorder to assay if the surface of the material was antimicrobial undernormal environmental exposure, the material was left open to theatmosphere for 1 month on an office table and lab bench to simulate realworld conditions. After illumination for 30 minutes under our standardconditions followed by colony counting, no colonies survived on anyplate, regardless of media or growth time.

Example 3

Retention of the PS within the NFC Matrix.

A formulation containing nanofibrillated cellulose with the additiveNeoCryl XK-98 (80:20, wt:wt based upon dry solids, respectively), termedNFC-NeoCryl 80/20 was generated. This formulation was observed to haveno significant loss of the PS when exposed to water.

Modulating the Spreading Characteristics of NFC Based Coatings.

The NFC-NeoCryl 80/20 formulation from TG-Ia shows satisfactoryspreading characteristics for application onto a number of substrates,including glass surfaces as well as aluminum, as shown below in TG-Ic.

Variation of the Photosensitizer within the NFC Matrix.

To enhance commercialization, a broad color palette for the NFC-NeoCryl80/20 can be advantageous (FIGS. 5A-5D). To demonstrate that theNFC-based materials can form a broad color palette for variousapplications, the following three photosensitizers were incorporated atabout 0.01-0.1 wt % into NFC-NeoCryl 80/20 on an aluminum foil backing:

(1) Green—TMPyP-Zn, a tetracationic porphyrin metallated with zinc toenhance photophysical properties.

(2) Red—The target BODIPY compound shown in FIG. 5D.

(3) Blue—Commercially available methylene blue (structure FIG. 5C).

The results are demonstrated in FIGS. 5A-5D.

Example 4

Antiviral Studies:

Vesicular stomatitis virus (VSV) was chosen for the initialinvestigation into the antiviral properties of NFC-NeoCryl 80/20. The0.1 wt % BODIPY material did not exhibit any antiviral activity againstVSV. However, upon illumination with visible light (400-700 nm) for 30minutes, both the 0.1 wt % TMPyP and 0.1 wt % methylene blue materialsdid show antiviral activity (FIG. 6). Notably, 0.1 wt % TMPyP inNFC-NeoCryl 80/20 showed a 91.5% reduction in infectivity, while 0.1 wt% methylene blue in NFC-NeoCryl 80/20 showed an impressive 99.95%reduction (over 2000-fold) (FIG. 6). No effect was seen in the absenceof illumination. Finally, it is important to note thatphotosensitizer-free NFC was equivalent to the material-free control,demonstrating that the viral particles do not stick to the material.

Example 5

Antibacterial Studies:

A multidrug-resistant strain of the Gram-negative bacteriumAcinetobacter baumannii (MDRAB) and a methicillin-resistant strain ofthe Gram-positive bacterium Staphylococcus aureus (MRSA) were chosen forthe initial investigation into the antibacterial properties ofNFC-NeoCryl 80/20 (FIGS. 7A-7B).

Methicillin-Resistant Staphylococcus aureus (MRSA):

0.1 wt % TMPyP in NFC-NeoCryl 80/20 killed 93% of the bacterium (FIG.7A) with about 30 minutes illumination with visible light (400-700 nm).Confirming the importance of the cationic charges for the inactivationof bacteria, a control porphyrin without ionic character showed nokilling under the same conditions. 0.1 wt % methylene blue inNFC-NeoCryl 80/20 showed about 58% killing.

Multidrug-Resistant Acinetobacter baumannii (MDRAB):

0.1 wt % TMPyP in NFC-NeoCryl 80/20 killed 72% of the bacterium (FIG.7B) with about 30 minutes illumination with visible light (400-700 nm).Confirming the importance of the cationic charges for the inactivationof bacteria, the control porphyrin without ionic character showed nokilling under the same conditions. 0.1 wt % methylene blue inNFC-NeoCryl 80/20 showed a higher level of killing at 86%.

1. A composition comprising: a natural polymer scaffold; and aphotosensitizer, wherein the photosensitizer is attached to the naturalpolymer scaffold.
 2. The composition of claim 1, wherein thephotosensitizer is attached to the natural polymer scaffold by acovalent bond.
 3. The composition of claim 1, wherein thephotosensitizer is attached to the natural polymer scaffold by anon-covalent bond.
 4. The composition of claim 3, wherein thenon-covalent bond is an electrostatic interaction.
 5. The composition ofclaim 1, wherein the photosensitizer is a positively chargedphotosensitizer.
 6. The composition of claim 5, wherein the positivelycharged photosensitizer is selected from the group consisting of:positively charged porphyrins, positively charged phthalocyanines,positively charged BODIPY based compounds, positively charged chlorins,positively charged bacteriochlorins, positively charged anthocyanins,positively charged rose Bengal, positively charged phenothiazinederivatives, and any permissible combinations thereof.
 7. Thecomposition of claim 1, wherein the photosensitizer is a negativelycharged photosensitizer.
 8. The composition of claim 7, wherein thewherein the negatively charged photosensitizer is selected from thegroup consisting of: negatively charged porphyrins (e.g.,5,10,15,20-tetrakis(4-sulphonatophenyl)porphyrin, 5,10,15,20-tetrakis(4carboxyphenyl)porphyrin-Pd(II)), negatively charged phthalocyanines(phthalocyanine tetrasulphonic acid), negatively charged BODIPY basedcompounds, negatively charged chlorins, negatively chargedbacteriochlorins, negatively charged anthocyanins, negatively chargedrose Bengal, negatively charged methylene blue and related phenothiazinederivatives, and any permissible combinations thereof.
 9. Thecomposition of claim 1, wherein the natural polymer scaffold is selectedfrom the group consisting of: nanofibrillated cellulose, nanocrystallinecellulose, cellulose fibers, starch, lignin, chitosan, nanofibrillatedchitosan, poly-glucosamine, and any permissible combinations thereof.10. The composition of claim 1, wherein the natural polymer scaffold isnegatively functionalized.
 11. The composition of claim 1, wherein thenatural polymer scaffold is positively functionalized.
 12. Thecomposition of 1-11 claim 1, further comprising a synthetic polymer. 13.The composition of claim 12, wherein the synthetic polymer ispolyacrylonitrile.
 14. The composition of claim 12, further comprising asecond photosensitizer, wherein the second photosensitizer is attachedto the synthetic polymer by a covalent or non-covalent interaction. 15.The composition of claim 1, wherein the composition forms a coating whenapplied to the surface of a material.
 16. The composition of claim 1,wherein the composition is antimicrobial.
 17. An object or a structurecomprising: a composition as in claim
 1. 18. The object or the structureof claim 17, wherein the composition forms a coating on a surface of theobject or the structure.
 19. The object or the structure of claim 17,wherein the composition is assimilated into the object or the structure.20. The object or the structure of claim 17, wherein the object or thestructure is a textile.
 21. The object or the structure of claim 17,wherein the object or the structure is a container or packagingmaterial.
 22. The object or the structure of claim 17, wherein theobject or the structure is a wall, ceiling, or floor.
 23. A method ofmaking the composition as in claim 1, the method comprising: mixing anatural polymer scaffold and a photosensitizer, wherein the step ofmixing forms a non-covalent or a covalent interaction between thenatural polymer scaffold and the photosensitizer.